Mutations in embB406 Are Associated with Low-Level Ethambutol Resistance in Canadian Mycobacterium tuberculosis Isolates

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2–5 μg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2–4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 μg/mL). These novel mutations may exacerbate variability in ethambutol resistance.


Introduction
Mycobacterium tuberculosis remains a priority pathogen for the World Health Organization (WHO) as the causative agent of tuberculosis and the second leading infectious cause of mortality worldwide [1].Globally, drug-resistant M. tuberculosis poses a serious risk to public health, as treatment options remain limited for resistant infections [1][2][3][4].In Canada, nearly 10% of all tuberculosis infections in 2022 were resistant to at least one first-line anti-tuberculosis drug [5].
Drug susceptibility testing (DST) for M. tuberculosis is performed using culture-based methods as the gold standard [6].Specifically, mycobacterial DST relies upon the proportion method and testing at a critical concentration, defined as the minimum drug concentration required to inhibit 99% of wild-type isolates of M. tuberculosis but does not inhibit strains that are resistant to antimicrobial therapy in vivo [6].These phenotypic assays are time-consuming due to the slow-growing nature of M. tuberculosis.Advances in molecular techniques such as PCR-based methods [7][8][9][10] and whole genome sequencing Antibiotics 2024, 13, 624 2 of 14 (WGS) [11][12][13] have enabled rapid detection of resistance-associated gene mutations.However, discordance between gold standard phenotypic DST and rapid molecular methods is reported for ethambutol [14][15][16].
Ethambutol is a bacteriostatic first-line anti-tuberculosis drug that targets arabinosyltransferases encoded by the embCAB operon, hindering the arabinogalactan biosynthetic pathway and thereby inhibiting mycobacterial cell wall synthesis [17].Mutations in the arabinosyltransferase-encoding embB gene, including embB306, embB406, and embB497, are known to convey ethambutol resistance in M. tuberculosis and are employed in diagnostic pipelines for rapidly predicting resistance to ethambutol [18][19][20].However, several studies have reported embB406 mutations in both ethambutol-resistant and susceptible isolates [15,16,[21][22][23].This discordance between genotypic and phenotypic DST at the current critical concentration questions the significance of embB406 mutations used for rapid molecular DST.
We hypothesize that embB mutations in codon 406 are associated with low-level ethambutol resistance which is undetectable by the current critical concentration (5 µg/mL) utilized for DST on the BACTEC™ MGIT™ 960 system.This study tabulates embB mutations found in Canadian M. tuberculosis isolates from 2002-2022, evaluates the impact of specific embB406 mutations on ethambutol resistance compared to the MGIT™ 960 method, and considers other embB mutations and novel mutations outside of embB which may exacerbate phenotypic variability.We found that mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 µg/mL) and that novel mutations may exacerbate variability in ethambutol resistance.
After tabulating all of the embB mutations found among isolates within the NRCM culture collection, the frequency of mutations in ethambutol-resistant and -sensitive isolates was used to determine the positive predictive value (PPV), specificity, and sensitivity for embB mutations using phenotypic DST as the gold standard.The detection of any embB mutation corresponded to a PPV of 45.7%, specificity of 97.2%, and sensitivity of 74.4%.An embB Gly406Asp mutation provided a PPV of 15.4%, specificity of 99.6%, and sensitivity of 2.3%.Finally, an embB Gly406Ala mutation corresponded with a PPV of 66.6%, specificity of 97.0%, and sensitivity of 2.3%.
Bold text indicates embB406 mutations.a S: sensitive; R: resistant.b MGIT960: BACTEC™ MGIT™ 960 system.c BACTEC460: BACTEC™ 460TB system.d Significance according to the 2023 WHO catalogue of mutations in M. tuberculosis complex and their association with drug resistance or susceptibility [19].e One isolate reported as resistant by routine phenotypic testing on the BACTEC™ 460TB system exhibited a sensitive result when subsequently tested on the BACTEC™ MGIT™ 960 system for confirmation of DST result.

Characteristics of embB406 Mutations
Mutations in embB codon 406 were the second most abundant embB mutation among ethambutol-resistant isolates in the culture collection, besides codon 306. Figure 1 shows the phylogenomic tree of all study isolates (n = 26) and ethambutol susceptibility based on molecular and phenotypic DST.
Of the 16 isolates with embB406 mutations, 13 held a Gly406Asp mutation and three exhibited a Gly406Ala mutation (Figure 1).All 16 isolates with embB406 mutations were predicted to be ethambutol-resistant by WGS-based molecular DST using MyKrobe Predictor.In contrast, phenotypic DST showed that just 25% (n = 4/16) isolates with an embB406 mutation were resistant to ethambutol at the critical concentration of 5 µg/mL.Of these four isolates, two had a Gly406Asp mutation while the other two harboured a Gly406Ala mutation.By mutation, 15.4% of all isolates with a Gly406Asp mutation (n = 2/13) and 66.6% of all isolates with a Gly406Ala mutation (n = 2/3) were resistant at 5 µg/mL.All isolates exhibited concordance between routine phenotypic DST results on the BACTEC™ 460TB system and the current BACTEC™ MGIT™ 960 system except Isolate 2, which was ethambutol-resistant on the BACTEC™ 460TB system while ethambutol-sensitive on the BACTEC™ MGIT™ 960 system.

Characteristics of embB406 Mutations
Mutations in embB codon 406 were the second most abundant embB mutation among ethambutol-resistant isolates in the culture collection, besides codon 306. Figure 1 shows the phylogenomic tree of all study isolates (n = 26) and ethambutol susceptibility based on molecular and phenotypic DST.Of the 16 isolates with embB406 mutations, 13 held a Gly406Asp mutation and three exhibited a Gly406Ala mutation (Figure 1).All 16 isolates with embB406 mutations were predicted to be ethambutol-resistant by WGS-based molecular DST using MyKrobe Predictor.In contrast, phenotypic DST showed that just 25% (n = 4/16) isolates with an embB406 mutation were resistant to ethambutol at the critical concentration of 5 µg/mL.Of these four isolates, two had a Gly406Asp mutation while the other two harboured a Conversely, 75% (n = 12/16) of isolates with an embB mutation were susceptible to ethambutol at 5 µg/mL, including 84.6% (n = 11/13) of isolates with a Gly406Asp mutation and 33.3% (n = 1/3) with a Gly406Ala mutation.DST below the critical concentration showed that all 12 isolates exhibited low-level ethambutol resistance (2-4 µg/mL), as described below.Finally, phenotypic DST for the 10 control pan-sensitive isolates showed that all had a wild-type embB406 gene locus and were sensitive to ethambutol at all tested concentrations (2-5 µg/mL).

embB406 and Phenotypic Susceptibility to Additional First-Line Drugs
Phenotypic susceptibility to first-line drugs rifampin, isoniazid, and pyrazinamide was also assessed by critical concentration on the BACTEC™ MGIT™ 960 system (Table 2).In total, 93.8% (n = 15/16) isolates exhibiting an embB mutation were resistant to at least one additional first-line drug.Six isolates were resistant to rifampin, 15 were resistant to isoniazid, and two were resistant to pyrazinamide.All isolates with rifampin resistance were also resistant to isoniazid (n = 6), and were classified as multi-drug resistant (MDR).Two of these isolates were resistant to all three drugs: rifampin, isoniazid, and pyrazinamide.Both MDR isolates with additional resistance to pyrazinamide harboured an embB Gly406Asp mutation (n = 2).MDR isolates without pyrazinamide resistance held either a Gly406Asp mutation (n = 2) or Gly406Ala mutation (n = 2).Nine isolates were resistant to isoniazid only and all featured mutations in embC Leu333Arg, embB Gly406Asp, and embR Gln258fs.A single isolate was mono-resistant to ethambutol and had an embB Gly406Ala mutation.

Discussion
Between years 2002-2022, 2794 M. tuberculosis isolates were screened for embB mutations in our laboratory.In total, ethambutol resistance was observed in 3.08% (n = 86/2794) isolates included in this study.A previous report on Canadian resistance to anti-tuberculosis drugs in 2021 recorded ethambutol resistance in 0.72% of M. tuberculosis isolates tested for anti-tuberculosis drug resistance (n = 11/1536) [24].The disparity between nationally reported rates for ethambutol resistance and our observations spanning 2002-2022 is likely due to the limitations of the culture collection included in this study.The NRCM does not receive all Canadian Mycobacterium tuberculosis complex (MTBC) isolates for DST, but rather receives MTBC from submitting Canadian laboratories with limited capacity for DST.As a result, this collection may not provide a complete picture of antimicrobial resistance among M. tuberculosis isolates in Canada.Outside of Canada, the rate of ethambutol resistance is reported to range from 0.3% to 14%, consistent with the present findings [25,26].
In total, embB mutations were observed in 5.01% (n = 140/2794) of M. tuberculosis isolates screened in this study, of which only 45.71% (n = 64/140) were phenotypically resistant to ethambutol.Notably, embB mutations were absent in 25.58% (n = 22/86) of ethambutol-resistant isolates in the present study.Accordingly, we show that the detection of an embB mutation in M. tuberculosis isolates corresponded to a PPV of 45.7%, specificity of 97.2%, and sensitivity of 74.4% for ethambutol phenotypic DST.These metrics show that single embB mutations provided low-confidence predictions of ethambutol resistance for this dataset.Other studies similarly report that embB mutations predicted ethambutol phenotypes with 81.3% sensitivity and 86.8% specificity, corroborating the low sensitivity of molecular DST employing embB mutations [16].
The 2023 WHO catalogue of MTBC mutations details molecular markers associated with drug resistance and classifies embB mutations according to a confidence grading system as "associated with resistance", "associated with interim resistance", "not associated with resistance", or "uncertain significance" [19,20].In the present study, the most abundant embB mutations "associated with resistance" occurred in codons 306 (n = 75), 406 (n = 16), and 497 (n = 5).Interestingly, discordance was recorded for each mutation observed at these loci, not only codon 406.
In total, 69.3% (n = 52/75) of the study isolates with a single mutation in embB306 were phenotypically resistant.By mutation, resistance was observed in 86.5% of isolates with a Met306Val mutation (n = 32/37), 52.8% of isolates with a Met306Ile mutation (n = 19/36), and 50% of isolates with a Met306Leu mutation (n = 1/2).In comparison, WHO reports the PPV for each mutation: resistance was observed in 82.6% of isolates with a Met306Val mutation (n = 3245/3930), 62.8% of isolates with a Met306Ile mutation (n = 1953/3112), and 74.4% of isolates with a Met306Leu mutation (n = 145/195) [19].Mutations in embB306 are well-studied and recognized as important molecular markers of ethambutol resistance [27].Allelic exchange studies have identified that single embB306 mutations cause an increase in ethambutol minimum inhibitory concentration (MIC) [28], however, there is some debate in the literature as to whether single embB306 mutations are sufficient to induce clinically significant ethambutol resistance [27][28][29].Here, we report that 30.7% (n = 23/75) of isolates with single embB306 mutations exhibit phenotypic susceptibility to ethambutol which is corroborated by several other studies that have identified embB306 mutations among ethambutol-susceptible isolates [21,29,30].These results and literature show a low to moderate capacity for embB306 mutations to predict ethambutol resistance using sequencing-based molecular DST.
A single resistance-associated mutation was observed in embB codon 497 (Gln497Arg) and 75% (n = 3/4) of isolates with this mutation were phenotypically resistant to ethambutol.WHO reports the PPV for Gln497Arg in which resistance was observed in 81.7% of isolates with this mutation (n = 999/1223) [19].Like embB306, allelic exchange experiments investigating embB497 suggest that these mutations may only make low-to-moderate contributions to ethambutol resistance [28].Again, these results and literature show moderate confidence in the ability of embB497 mutations to predict ethambutol resistance.
Other embB mutations "associated with resistance" were observed in singular ethambutol-resistant isolates in this study: Tyr319Ser, Asp328Tyr, Asp354Ala.Mutations were also observed that are "not associated with resistance" or have "uncertain significance" for ethambutol resistance according to the 2023 WHO mutation catalogue [19].One mutation, Gly378Ala, is "not associated with resistance" and was observed in 37 isolates, of which only a single isolate was ethambutol-resistant. Four mutations categorized with "uncertain significance", including Ile226Val, Ala313Val, Ala386Ser and Gln497Pro were observed in four isolates, all of which were phenotypically sensitive to ethambutol.Finally, embB mutation Tyr333Asn is not documented in the WHO mutation catalogue and was observed a single ethambutol-sensitive isolate.
The present study specifically evaluates ethambutol resistance among M. tuberculosis isolates with embB406 mutations due to the accumulating reports of sensitivity among isolates with this mutation [21][22][23][31][32][33][34].Indeed, only 25% (n = 4/16) of isolates with embB406 mutations in the NRCM culture collection tested as ethambutol-resistant at the current critical concentration using the BACTEC™ MGIT™ 960 system.By mutation, 15.4% (n = 2/13) of isolates with a Gly406Asp mutation were reported as resistant and 66.6% (n = 2/3) of isolates with a Gly406Ala mutation were resistant.These data correspond to a PPV of 15.4%, specificity of 99.6%, and sensitivity of 2.3% for Gly406Asp and a PPV of 66.6%, specificity of 97.0%, and sensitivity of 2.3% for Gly406Ala.In comparison, WHO reports a PPV of 62.2%, specificity of 99.4%, and sensitivity of 3.5% for Gly406Ala and PPV of 56.4%, specificity of 99.4%, and sensitivity of 2.9% for Gly406Asp [19].Our results in a limited number of isolates show that embB406 mutations are a low-confidence predictor of ethambutol resistance, and are corroborated by the WHO's reports of low sensitivity and predictive values for these mutations.
Here, we investigated discordance between molecular methods and phenotypic DST for ethambutol on the BACTEC™ MGIT™ 960 system.This discordance is not isolated to the BACTEC™ MGIT™ 960 system but is also reported for other WHO-and Clinical and Laboratory Standards Institute (CLSI)-endorsed susceptibility testing methods [6,19,20] and are summarized in Table 3. DST by agar proportion on solid media employs different critical concentrations for ethambutol depending on media type: 2 µg/mL by Löwenstein-Jensen (LJ), 5 µg/mL by Middlebrook 7H10 agar, and 7.5 µg/mL by Middlebrook 7H11 agar [6].Few studies describe concordance between embB406 mutations and phenotypic DST using the agar proportion method [31,33].Several report embB406 mutations among both resistant and susceptible isolates by agar proportion on LJ media [16,23,32,34,35].Park et al. (2012) describe moderate ethambutol resistance (0.88-4 µg/mL) among M. tuberculosis isolates with embB406 mutations [23].Others employing agar proportion DST have suggested that ethambutol resistance is multigenic and that embB406 mutations alone are insufficient for the development of resistance [21,28].The BACTEC™ 460TB and BACTEC™ MGIT™ 960 systems employ agar proportionequivalent ethambutol critical concentrations of 2.5 µg/mL and 5 µg/mL, respectively [6,40,41].The adaptation of critical concentrations from agar proportion methods to the BACTEC™ 460TB system and the BACTEC™ MGIT™ 960 system shortly thereafter, has been criticized for poor concordance and standardization [15,42].In the present study, 18 isolates with embB mutations associated with resistance were tested as phenotypically resistant by BACTEC™ 460TB (100% concordance between molecular and phenotypic DST).In contrast, 45 out of 80 isolates harbouring an embB mutation associated with resistance tested as resistant on the BACTEC™ MGIT™ 960 system (56.3%concordance between molecular and phenotypic DST).This study also detected one isolate with a Gly406Ala embB mutation that tested as ethambutol-sensitive on the BACTEC™ 460TB but resistant on the BACTEC™ MGIT™ 960 system.Several others also describe ethambutol-susceptible isolates with embB406 mutations [15,16,21,36].Bwalya et al. (2022) and Christianson et al. (2014) report low-level ethambutol resistance among isolates with embB406 mutations [15,22].These observations are often accompanied by a recommendation that the BACTEC™ MGIT™ 960 critical concentration may be lowered to ensure that resistance at or near the 5 µg/mL critical concentration is detected [15].
Previous studies have suggested that common mechanisms of resistance exist between isoniazid and ethambutol given their shared drug target [43] and synergistic activity [44].Accordingly, we briefly investigated the susceptibility of isolates with embB406 mutations to the remaining three first-line anti-tuberculosis drugs: isoniazid, rifampin, and pyrazinamide.Ethambutol mono-resistance, while rare in literature, was observed in a single isolate with an embB Gly406Ala mutation.Isoniazid resistance was observed in 93.75% (n = 15/16) of isolates harbouring a mutation in embB406.Similarly, another study reported simultaneous resistance to ethambutol and isoniazid in 85.18% of M. tuberculosis isolates that underwent phenotypic DST [45].However, additional studies are required to solidify a clear mechanistic linkage between resistance to ethambutol and other antimicrobial agents.
Expanding the scope of this examination, we investigated other genes within the embCAB operon, including the operon's regulator embR.As a result, it was found that nine isolates exhibited identical mutations in embC (Leu333Arg) and embR (Gln258fs) which have not been previously described, in addition to the embB Gly406Asp mutation.All nine isolates with this mutation profile were sensitive at 5 µg/mL ethambutol but exhibited low-level resistance undetectable at the critical concentration.Specifically, 11.1% (n = 1/9) were resistant at 4 µg/mL, 33.3% (n = 3/9) were resistant at 3 µg/mL, and 55.6% (n = 5/9) were resistant at 2 µg/mL.Notably, the embR frameshift mutation occurs within the forkhead-associated (FHA) domain of the operon's regulator.As the FHA domain is critical for phosphorylation and activation of embR [46], we predict that this novel and disruptive frameshift mutation may impact embCAB expression and cause variability in resistance.Further investigation of these mutations is required to isolate their impact on ethambutol resistance.
While this study included all strains with molecular and phenotypic DST in the Canadian National Reference Centre for Mycobacteriology culture collection, the limited number of strains with embB406 mutations included in this study must be acknowledged.Even so, our results corroborate a wealth of studies calling for improved concordance between molecular and phenotypic DST for ethambutol.The results herein illustrate that embB406 mutations are low-confidence mutations associated with low-level ethambutol resistance.It is possible that mutations outside of embB, including embR, may promote variability in ethambutol resistance.Molecular DST is an invaluable tool to rapidly inform patient treatment regimens while gold culture methods remain time-consuming with long turnaround times.However, discordance between molecular and phenotypic methods must be acknowledged with caution.A greater understanding of the impact of embB mutations on ethambutol resistance as well as the clinical significance of low-level resistance is required to inform the improvement of DST.

Study Isolates
The culture collection housed within the NRCM spanning 2002-2022 (n = 2794) was screened for M. tuberculosis isolates containing embB mutations by routine sequencing of the embB gene by WGS and Sanger sequencing.Screening identified 16 isolates harbouring an embB406 mutation, for which WGS-based SNP analysis and extended phenotypic ethambutol susceptibility testing were performed (described below).Pan-sensitive isolates lacking embB mutations (n = 10) and M. tuberculosis strain H37Rv ATCC 27294 were included in SNP analysis and extended phenotypic ethambutol susceptibility testing as control isolates.
SNP analysis was performed for strains with embB406 mutations and pan-sensitive control strains.Using Snippy [55] (https://github.com/tseemann/snippy,accessed on 29 April 2024).sequencing reads were assembled and compared to an annotated M. tuberculosis H37Rv reference genome (AL_123456.3) for SNP calling.The quality of the alignment used for SNP detection was determined with QualiMap2 [56] (version 2.2.2d).Minimum quality thresholds for alignments included genome coverage of 30, 90% of reads mapping to the reference genome, a mapping quality of 58.5, and GC content of 65 ± 1%.Alignments were visualized in Geneious [49] (version 11.0.12).SNVPhyl [57] (version 1.2.3) was used for the phylogenomic analysis of all strains in the study, utilizing the M. tuberculosis H37Rv genome as a reference (NC_000962.3).Microreact [58] (https: //microreact.org,accessed on 29 April 2024) was used to visualize phylogenic trees.

Figure 2 .
Figure 2. Mutations and Codon Changes Found in the embCAB Operon of M. tuberculosis Isolates.Linear gene (green) and protein domain (key) map of single nucleotide polymorphisms in the

Figure 2 .
Figure 2. Mutations and Codon Changes Found in the embCAB Operon of M. tuberculosis Isolates.Linear gene (green) and protein domain (key) map of single nucleotide polymorphisms in the embCAB operon.Identity and position of base pair (bp) and amino acid (aa) substitutions are shown.n indicates the total number of ethambutol-resistant isolates harbouring a given mutation.The black arrows represent mutations found in embB406 mutants only.The white arrows indicate mutations found in both embB406 mutants and pan-susceptible control isolates.

Table 1 .
List of embB Mutations and Corresponding Phenotypic Susceptibility Testing Data for Study Isolates from Years 2002-2022.

Table 2 .
A Summary of Drug Resistance and embCAB Mutation Profiles of M. tuberculosis Isolates.